View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Radboud Repository PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a postprint version which may differ from the publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/144055 Please be advised that this information was generated on 2017-12-05 and may be subject to change. Isolation and identification of 4-α-rhamnosyloxy benzyl glucosinolate in Noccaea caerulescens showing intraspecific variation Rob M. de Graaf1,9*, Sebastian Krosse1, Ad E.M. Swolfs2, Esra te Brinke3, Nadine Prill4, Roosa Leimu4, Peter M. van Galen5, Yanli Wang6, Mark G.M. Aarts6, Nicole M. van Dam1,7,8 1.Molecular Interaction Ecology, Institute of Water and Wetland Research (IWWR), Radboud University Nijmegen, PO Box 9010, 6500 GL Nijmegen, The Netherlands 2. Synthetic Organic Chemistry, Institute for Molecules and Materials (IMM), Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands 3. Physical Organic Chemistry, Institute for Molecules and Materials (IMM), Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands 4. Department of Plant Sciences, University of Oxford, South Parks Road, OX1 3RB, UK 5. Bio-organic Chemistry, Institute for Molecules and Materials(IMM), Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands 6. Laboratory of Genetics, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands 7. German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, D- 04103 Leipzig, Germany 8. Friedrich Schiller University Jena, Institute of Ecology, Dornburger-Str. 159, 07743 Jena, Gemany 9. Deparment of Microbiology, Institute of Water and Wetland Research (IWWR), Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands * Corresponding author, Tel.: +31 243652569. , Fax.: +31 243553450. E-mail address: [email protected] (R.M. de Graaf) 1 Abstract Glucosinolates are secondary plant compounds typically found in members of the Brassicaceae and a few other plant families. Usually each plant species contains a specific subset of the ~130 different glucosinolates identified to date. However, intraspecific variation in glucosinolate profiles is commonly found. Sinalbin (4-hydroxybenzyl glucosinolate) so far has been identified as the main glucosinolate of the heavy metal accumulating plant species Noccaea caerulescens (Brassicaceae). However, a screening of 13 N. caerulescens populations revealed that in 10 populations a structurally related glucosinolate was found as the major component. Based on Nuclear Magnetic Resonance (NMR) and mass spectrometry analyses of the intact glucosinolate as well as of the products formed after enzymatic conversion by sulfatase or myrosinase, this compound was identified as 4-α-rhamnosyloxy benzyl glucosinolate (glucomoringin). So far, glucomoringin had only been reported as the main glucosinolate of Moringa spp. (Moringaceae) which are tropical tree species. There was no apparent relation between the level of soil pollution at the location of origin, and the presence of glucomoringin. The isothiocyanate that is formed after conversion of glucomoringin is a potent antimicrobial and antitumor agent. It has yet to be established whether glucomoringin or its breakdown product have an added benefit to the plant in its natural habitat. Keywords: glucosinolates, intraspecific variation, chemotypes, Thlaspi, Noccaea caerulescens, populations, isothiocyanate 2 1. Introduction Glucosinolates are a large class of sulfur and nitrogen containing plant secondary metabolites that are produced by most plant species belonging to the order Brassicales (Agerbirk and Olsen, 2012; Fahey, 2005). The core of every glucosinolate molecule is formed by a thiohydroximate group carrying two residues; an S-linked beta-glucopyranosyl moiety, and an O-linked sulfate residue (Agerbirk and Olsen, 2012). Additionally, each glucosinolate is characterized by a variable R-group. This R-group is synthesized from different amino-acids, such as leucine, valine, tryptophan and phenylalanine, and is used to further sub-divide glucosinolates into different structural groups, often referred to as aliphatic, indole and aromatic, or benzylic, glucosinolates (Agerbirk and Olsen, 2012; Fahey et al., 2001). In a recent review Agerbirk and Olsen (2012) reported that around 132 different natural glucosinolates are identified to date. For a part, the distribution of different types of glucosinolates is phylogenetically constrained due to the presence or absence of certain biosynthetic genes in the different branches of the Brassicales’ evolutionary tree. For example, it was stated that glucosinolates with glycosylated R-groups appear to be limited to the Resedaceae and Moringaceae (Fahey et al., 2001). Also within plant families there is substantial variation in the ability to produce certain glucosinolates, due to evolutionary events including deletions and small-scale or whole genome duplications contributing to loss or gain of biosynthetic genes (Bekaert et al., 2012). As a consequence, each plant species has its own typical glucosinolate profile which may contain up to 37 different glucosinolates (Kliebenstein et al., 2001). The glucosinolates, together with the β-thioglucosidase myrosinase, form a specific defence system against herbivores (Ahuja et al., 2010; Hopkins et al., 2009). In intact plants, myrosinase enzymes and glucosinolates are stored separately. As soon as plants are damaged by herbivore feeding or by artificial wounding, myrosinase and glucosinolates are mixed and react. Depending on the pH, and on the presence or absence of specific modifier proteins, such as nitrile specifier or epithionitrile specifier proteins, the glucosinolates are quickly converted to nitriles, epithionitriles or isothiocyanates (Agerbirk and Olsen, 2012; Kissen and Bones, 2009; Kissen et al., 2009; Wittstock and Halkier, 2002). This two-component 3 defence system has been coined as the “the mustard oil bomb” (Kissen et al., 2009; Ratzka et al., 2002). Especially the isothiocyanates, which also give cabbages and mustards their pungent flavour, are toxic or deterrent to a wide range of herbivores and pathogens (Brown and Morra, 1997; Hopkins et al., 2009; Park et al., 2013). Another property of isothiocyanates is that they contribute to human health. They can function as cancer preventing agents and can inhibit growth of bacteria such as Helicobacter pylori, which is the causal agent of gastritis (Fahey et al., 2002; Halkier and Gershenzon, 2006). Especially sulforaphane, the breakdown product of glucoraphanin (4-methylsulfinylbutyl glucosinolate), which is commonly found in high concentrations in broccoli, has been extensively studied and promoted because of its beneficial health effects (Fahey et al., 2002; Verkerk et al., 2009). Similarly, in many tropical countries extracts of the leaves, seeds and roots of Moringa tree species, belonging to the family Moringaceae, are used for a large range of medical uses (Eilert et al., 1981; Kumar et al., 2010). The main glucosinolate found in this tree is 4-(α-L-rhamnosyloxy) benzyl glucosinolate or glucomoringin, a rhamnose derivative of sinalbin (Amaglo et al., 2010; Bennett et al., 2003; Gueyrard et al., 2010; Mekonnen and Drager, 2003). The isothiocyanate of glucomoringin is a biologically very active compound that is reported to have a beneficial effect on a broad spectrum of human diseases, ranging from bacterial infections to cancer (Fahey, 2005; Faizi et al., 1994; Ragasa et al., 2012). Until now glucomoringin and structurally related O-glycosylated glucosinolates had only been identified in members of the Moringaceae and Resedaceae. Here we report the identification of glucomoringin and its isothiocyanate breakdown product as isolated from Noccaea caerulescens (J.Presl & C.Presl) F.K.Mey., synonym. Thlaspi caerulescens J.Presl & C.Presl, which is a member of the Brassicaceae. N. caerulescens is extensively studied because of its capacity to accumulate large quantities of heavy metals, such as cadmium and zinc, when grown on polluted soils (Assuncao et al., 2003; Leitenmaier and Kupper, 2013). For this reason, the species is used as a phytoremediator to clean soils contaminated with heavy metals due to mining or industrial activities. Several studies were performed to assess the influence of cadmium (Cd) and zinc (Zn) exposure and accumulation on the glucosinolate content of metal hyperaccumulators. Sinalbin and sinigrin, plus several other aliphatic, benzylic and indoyl glucosinolates 4 were identified in N. caerulescens (Tolra et al., 2000) or in related species (Noccaea praecox, Thlaspi arvense) (Pongrac et al., 2008; Tolra et al., 2006). However, glucomoringin was never reported to be present ( Asad et al., 2013; Tolra et al., 2000). For this reason, we isolated the main glucosinolate peak of our N. caerulescens extracts and applied advanced NMR and mass spectrometry analyses (Agerbirk and Olsen, 2012; Bennett et al., 2006) to accurately identify the molecular structure of this glucosinolate. Furthermore, we analysed seeds and leaves from 13 different populations in Europe to study natural variation in glucosinolate profiles in this species. 5 2. Results and Discussion Desulfoglucosinolate extracts of N. caerulescens were first analysed by HPLC-PDA (229